Method for treating a pulmonary disease state in mammals by up regulating indigenous in vivo levels of inflammatory agents in mammalian cells

ABSTRACT

The present invention provides novel methods for treating a pulmonary disease state in mammals by up or down regulating indigenous in vivo levels of an inflammatory agent in mammalian cells comprising contacting the mammalian cells with a therapeutically effective amount of an inflammatory regulator, wherein the inflammatory agent is selected from the group consisting of cytokines, transforming growth factor-β, elastase, and white blood cells, and wherein the inflammatory regulator is selected from the group consisting of pyruvates and pyruvate precursors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 11/890,911filed on Aug. 8, 2007, now U.S. Pat. No. 8,114,907

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention provides novel methods for treating a pulmonarydisease state in mammals by up or down regulating indigenous in vivolevels of an inflammatory agent in mammalian cells comprising contactingthe mammalian cells with a therapeutically effective amount of aninflammatory regulator, wherein the inflammatory agent is selected fromthe group consisting of cytokines, transforming growth factory,elastase, and white blood cells, and wherein the inflammatory regulatoris selected from the group consisting of pyruvates and pyruvateprecursors.

2. Background of the Invention

The disclosures referred to herein to illustrate the background of theinvention and to provide additional detail with respect to its practiceare incorporated herein by reference and, for convenience, arereferenced in the following text and respectively grouped in theappended bibliography.

Inflammatory agents are produced by a wide variety of body cells and arenatural proteins produced by the cells of the immune system of mostvertebrates in response to challenges by foreign agents such as viruses,bacteria, parasites, and tumor cells (1).

Cytokines are a group of proteins and peptides that are used inorganisms as signaling compounds and are used to allow one cell tocommunicate with another. The cytokine family consists mainly of smallerwater-soluble proteins and glycoproteins. Cytokines are released by manytypes of cells, principally activated lymphocytes, and macrophages butalso endothelium, epithelium and connective tissue. They areparticularly important in both innate and adaptive immune responses. Dueto their central role in the immune system, cytokines are involved in avariety of immunological, inflammatory and infectious diseases.

Interleukins (ILs) are a group of inflammatory cytokines that were firstseen to be expressed by white blood cells. Interleukins are produced bya wide variety of bodily cells including endothelial cells andmacrophages. The family of interleukins includes IL-1 to IL-33. Thefunction of the immune system depends in a large part on interleukins,and rare deficiencies of a number of them have been described, allfeaturing autoimmune diseases or immune deficiency.

Interferons (IFNs) belong to a large class of glycoproteins and arecytokines. Interferons are natural proteins produced by the cells of theimmune system of most vertebrates in response to challenges by foreignagents such as viruses, bacteria, parasites and tumor cells. Interferonsassist the immune response by inhibiting viral replication within othercells of the body.

Tumor necrosis factor (TNF) is a cytokine involved in systemicinflammation and is a member of a group of cytokines that all stimulatethe acute phase reaction. Tumor necrosis factor causes apoptotic celldeath, cellular proliferation, differentiation, inflammation,tumorigenesis, and viral replication. Tumor necrosis factor's primaryrole is in the regulation of immune cells. Dysregulation and, inparticular, overproduction of tumor necrosis factor have been implicatedin a variety of human diseases, as well as cancer

Chemokines are a family of small cytokines, or proteins that areclassified according to shared structural characteristics such as smallsize (they are all approximately 8-10 kilo Daltons in size), and thepresence of four cysteine residues in conserved locations that are keyto forming their 3-dimensional shape. Chemokines have the ability toinduce directed chemotaxis in nearby responsive cells (chemotacticcytokines). Some chemokines are considered pro-inflammatory and can beinduced during an immune response to promote cells of the immune systemto a site of infection, while others are considered homeostatic and areinvolved in controlling the migration of cells during normal processesof tissue maintenance or development. Chemokines exert their biologicaleffects by interacting with G protein-linked transmembrane receptorscalled chemokine receptors that are selectively found on the surfaces oftheir target cells.

Cytokines and chemokines have the ability to stimulate leukocytemovement and play an important role in inflammation. Cytokines caninfluence the synthesis of other cytokines and chemokines. Cytokines canalso stimulate cell proliferation acting as growth factors. Cytokinesthat regulate lymphocyte activation, growth and differentiation includeIL-2, IL-4, IL-10, and TNF-β. Cytokines involved with natural immunity,inflammation, include TNF-α, IL-1, INF-α, INF-β, and IL-6. Cytokinesthat activate inflammatory cells like macrophages include IFN-γ, TNF-α,TNF-β, IL-5, IL-10, IL-12, and IL-8. Cytokines that stimulatehemopoiesis and mediate immature leukocyte growth and differentiationinclude IL-3, IL-7, c-kit ligand, granulocyte-macrophage, granulocytecolony-stimulating factor (G-CSF), and stem cell factor. Granulocytecolony-stimulating factor is a glycoprotein, growth factor or cytokineproduced by a number of different tissues to stimulate the bone marrowto produce granulocytes and stem cells. Granulocyte colony-stimulatingfactor then stimulates the bone marrow to pulse them out of the marrowinto the blood.

IL-8 is responsible in attracting white blood cells to the site ofinfection. The major cytokines that mediate inflammation are IL-1, IL-8,and TNF (α and β). IL-1 and TNF-α are produced by activated macrophages.Their secretion can be stimulated by infections, endotoxins, immunecomplexes, toxins, physical injury, and a variety of inflammatoryprocesses. Their most important actions in inflammation are their effecton endothelium, leukocytes, and fibroblasts and induction of thesystemic acute phase reactions. TNF also cause aggregation and primingof neutrophils, leading to a release of proteolytic enzymes, thuscontributing to tissue damage. TNF-α, IL-1, and IL-6 also induce thesystemic acute phase responses associated with infection, or injury,including fever, loss of appetite, the production of slow wave sleep,release of neutrophils into circulation, release of hormones,hemodynamic effects of septic shock, hypotension, decrease in vascularresistance, increased heart rate, and decrease in blood pH.

An excess of inflammatory agents can increase the production of oxygenradicals, including superoxide anions and hydrogen peroxide, producedduring the inflammatory phase of an injury, which will destroy healthytissue surrounding the site and will mitigate the positivebronchodilation effect of nitric oxide (26). Oxygen radicals can alsoinitiate lipid peroxidation employing arachidonic acid as a substrateproducing prostaglandins and leukotrienes. Hydrogen peroxide (H₂O₂) caninduce arachidonic acid metabolism in alveolar macrophages (17,26).Oxygen radicals also produce 8-isoprostanes, which are potent renal andpulmonary artery vasoconstrictors, bronchoconstrictors, and induceairflow obstructions (26, 27). Because oxygen radicals contribute to theinstability of nitric oxide, the addition of superoxide dismutase (SOD)or catalase (15) or Vitamin E (28) protect nitric oxide to produce itsdesired bronchodilation (2). Hydrogen peroxide is elevated in patientswith chronic obstructive pulmonary disease (COPD), asthma, and AcuteRespiratory Distress Syndrome (ARDS) (26). A study in 28 patients showeda significant correlation between oxygen radical generation in whiteblood cell count (WBC) and the degree of bronchial hyperreactivity invivo in nonallergic patients (18). Thus the ability of pyruvate toregulate inflammation, and inflammatory agents, which can increase thesynthesis of oxygen radicals, should reduce the production of oxygenradicals when needed.

Sodium pyruvate is an antioxidant that reacts directly with oxygenradicals to neutralize them. In macrophages, and other cell lines,sodium pyruvate regulates the level of oxygen radicals by acting as anantioxidant and also increases the synthesis of nitric oxide (9). It canspecifically lower the overproduction of superoxide anions. Sodiumpyruvate also increases cellular levels of glutathione, a major cellularantioxidant (12). It was recently discovered that glutathione is reduceddramatically in antigen-induced asthmatic patients (13) and inhaledglutathione does not readily enter cells. Pyruvate does enter all cellsvia a transport system and can also cross the blood brain barrier.Excess sodium pyruvate beyond that needed to neutralize oxygen radicalswill enter the bronchial and lung cells. All cells have a transportsystem that allow cells to concentrate pyruvate at higher concentrationsthan serum levels. In the cell, pyruvate raises the pH level, increaseslevels of ATP, decreasing levels of ADP and cAMP, and increases levelsof GTP, while decreasing levels of cgMP. Nitric oxide (NO) acts in theopposite mode by increasing levels of cGMP and ADP, and requires anacidic pH range in which to work (19). While the above therapeuticcompositions and methods are reported to inhibit the production ofreactive oxygen intermediates, like hydrogen peroxide or peroxynitrite,none of the disclosures describe methods for treating a pulmonarydisease state in mammals by regulating indigenous in vivo levels ofinflammatory agents in mammalian cells.

U.S. Pat. No. 6,063,407 (Zapol et al.) discloses methods of treating,inhibiting or preventing vascular thrombosis or arterial restenosis in amammal. The methods include causing the mammal to inhale atherapeutically effective concentration of gaseous nitric oxide. Theinhaled nitric oxide may further comprise compounds that potentiate thebeneficial effects of inhaled nitric oxide and antithrombotic agentsthat complement or supplement the beneficial effects of inhaled nitricoxide.

U.S. Pat. No. 6,020,308 (Dewhirst et al.) discloses the use of aninhibitor of nitric oxide activity, such as a nitric oxide scavenger ora nitric oxide synthase inhibitor, as an adjunct to treatment ofinappropriate tissue vascularization disorders.

U.S. Pat. No. 5,891,459 (Cooke et al.) discloses the maintenance orimprovement of vascular function and structure by long termadministration of physiologically acceptable compounds, such asL-arginine, L-lysine, physiologically acceptable salts thereof, andpolypeptide precursors thereof, which enhance the level of endogenousnitric oxide or other intermediates in the nitric oxide inducedrelaxation pathway in the host. The method further comprises theadministration of other compounds, such as B6, folate, B12, or anantioxidant, which provide for short-term enhancement of nitric oxide.

U.S. Pat. No. 5,873,359 (Zapol et al.) discloses a method for treatingor preventing bronchoconstriction or reversible pulmonaryvasoconstriction in a mammal, which method includes causing the mammalto inhale a therapeutically effective concentration of gaseous nitricoxide or a therapeutically effective amount of a nitric oxide releasingcompound and an inhaler device containing nitric oxide gas and/or anitric oxide releasing compound.

U.S. Pat. No. 5,767,160 (Kaesemeyer) discloses a therapeutic mixturecomprising L-arginine and an agonist of nitric oxide synthase, such asnitroglycerin for the treatment of diseases related to vasoconstriction.The vasoconstriction is relieved by stimulating the constitutive form ofnitric oxide synthase (cNOS) to produce native nitric oxide. The nativenitric oxide has superior beneficial effect when compared to exogenousnitric oxide produced by a L-arginine independent pathway in terms ofthe ability to reduce clinical endpoints and mortality.

U.S. Pat. No. 5,543,430 (Kaesemeyer) discloses a therapeutic mixturecomprising a mixture of L-arginine and an agonist of nitric oxidesynthase such as nitroglycerin for the treatment of diseases related tovasoconstriction. The vasoconstriction is relieved by stimulating theconstitutive form of nitric oxide synthase to produce native nitricoxide. The native nitric oxide has superior beneficial effect whencompared to exogenous nitric oxide produced by a L-arginine independentpathway in terms of the ability to reduce clinical endpoints andmortality.

U.S. Pat. No. 5,428,070 (Cooke et al.) discloses a method for treatingatherogenesis and restenosis by long-term administration ofphysiologically acceptable compounds, which enhance the level ofendogenous nitric oxide in the host. Alternatively, or in combination,other compounds may be administered which provide for short-termenhancement of nitric oxide, either directly or by physiologicalprocesses. In addition, cells may be genetically engineered to provide acomponent in the synthetic pathway to nitric oxide, so as drive theprocess to enhance nitric oxide concentration, particularly inconjunction with the administration of a nitric oxide precursor.

U.S. Pat. No. 5,286,739 (Kilbourn et al.) discloses an anti-hypotensiveformulation comprising a mixture of amino acids, which is essentiallyarginine free or low in arginine (less than about 0.1%, most preferably,about 0.01%). The formulation may include ornithine, citrulline, orboth. A method for prophylaxis and treatment of systemic hypotension inan animal is also provided. A method for treating hypotension caused bynitric oxide synthesis through administering a low or essentiallyarginine free parenteral formulation to an animal, so as to reduce oreliminate nitric oxide synthesis is described. A method for treating ananimal in septic shock is also disclosed, comprising administering tothe animal an anti-hypotensive formulation comprising a mixture of aminoacids, which is essentially arginine free. Prophylaxis or treatment ofsystemic hypotension, particularly that hypotension incident tochemotherapeutic treatment with biologic response modifiers, such astumor necrosis factor or interleukin-1 or 2, may be accomplished throughthe administration of the defined anti-hypotensive formulations untilphysiologically acceptable systolic blood pressure levels are achievedin the animal. Treatment of an animal for septic shock induced byendotoxin may also be accomplished by administering to the animal thearginine free formulations.

U.S. Pat. No. 5,217,997 (Levere et al.) discloses a method for treatinga high vascular resistance disorder in a mammal by administering to amammalian organism in need of such treatment a sufficient amount ofL-arginine or pharmaceutically acceptable salt thereof to treat a highvascular resistance disorder. The L-arginine is typically administeredin the range of about 1 mg to 1500 mg per day. High vascular resistancedisorders include hypertension, primary or secondary vasospasm, anginapectoris, cerebral ischemia and preeclampsia. Also disclosed is a methodfor preventing or treating bronchial asthma in a mammal by administeringto a mammalian organism in need of such prevention or treatment asufficient amount of L-arginine to prevent or treat bronchial asthma.

U.S. Pat. No. 5,158,883 (Griffith) discloses pharmaceutically purephysiologically active NG-aminoarginine (i.e., the L or D, L form), orpharmaceutically acceptable salts thereof, administered in a nitricoxide synthesis inhibiting amount to a subject in need of suchinhibition (e.g., a subject with low blood pressure or needingimmunosuppressive effect) or added to a medium containing isolatedorgans, intact cells, cell homogenates or tissue homogenates in anamount sufficient to inhibit nitric oxide formation to elucide orcontrol the biosynthesis, metabolism or physiological role of nitricoxide.

U.S. Pat. Nos. 5,798,388, 5,939,459, and 5,952,384 (Katz) pertain tomethods for treating various disease states in mammals caused bymammalian cells involved in the inflammatory response and compositionsuseful in the method. The method comprises contacting the mammaliancells participating in the inflammatory response with an inflammatorymediator. The inflammatory mediator is present in an amount capable ofreducing the undesired inflammatory response and is an antioxidant. Thepreferred inflammatory mediator is a pyruvate. Katz discloses thetreatment of airway diseases of the lungs such as bronchial asthma,acute bronchitis, emphysema, chronic obstructive emphysema,centrilobular emphysema, panacinar emphysema, chronic obstructivebronchitis, reactive airway disease, cystic fibrosis, bronchiectasis,acquired bronchiectasis, kartaagener's syndrome; atelectasis, acuteatelectasis, chronic atelectasis, pneumonia, essential thrombocytopenia,legionnaires disease, psittacosis, fibrogenic dust disease, diseases dueto organic dust, diseases due to irritant gases and chemicals,hypersensitivity diseases of the lung, idiopathic infiltrative diseasesof the lungs and the like by inhaling pyruvate containing compositions.

U.S. Pat. No. 5,296,370 (Martin et al.) discloses therapeuticcompositions for preventing and reducing injury to mammalian cells andincreasing the resuscitation rate of injured mammalian cells. Thetherapeutic composition comprises (a) pyruvate selected from the groupconsisting of pyruvic acid, pharmaceutically acceptable salts of pyruvicacid, and mixtures thereof, (b) an antioxidant, and (c) a mixture ofsaturated and unsaturated fatty acids wherein the fatty acids are thosefatty acids required for the resuscitation of injured mammalian cells.

U.S. Pat. No. 6,689,810 (Martin) discloses a therapeutic composition fortreating pulmonary diseases states in mammals by altering indigenous invivo levels of nitric oxide. The therapeutic composition consists ofpyruvates, pyruvate precursors, α-keto acids having four or more carbonatoms, precursors of α-keto acids having four or more carbons, and thesalts thereof.

U.S. Pat. No. 7,122,578 (Martin) discloses a therapeutic composition fortreating topical diseases states and injuries in mammals involvinginjuries, which cause pain, erythema, swelling, crusting, ischemia,scarring, and excess white blood cell infiltration. The method involvesthe use of α-keto acids to suppress inflammation.

WO 2006/086643 (Martin) discloses a non-pulmonary treatment of mammaliandiseases and injuries caused by the over-expression of peroxynitrite.

While the above therapeutic compositions and methods are reported toinhibit the production of reactive oxygen intermediates, such ashydrogen peroxide, peroxynitrite or nitric oxide, none of thedisclosures describe a method for treating a pulmonary disease state inmammals by altering indigenous in vivo levels of inflammatory agents.

SUMMARY OF THE INVENTION

The present invention provides novel methods for treating a pulmonarydisease state in mammals by up or down regulating indigenous in vivolevels of an inflammatory agent in mammalian cells comprising contactingthe mammalian cells with a therapeutically effective amount of aninflammatory regulator, wherein the inflammatory agent is selected fromthe group consisting of cytokines, transforming growth factor-β,elastase, and white blood cells, and wherein the inflammatory regulatoris selected from the group consisting of pyruvates and pyruvateprecursors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating individual sputum total protein levelsbefore and after study drug inhalation. Slash marks represent the medianlevel.

FIG. 2 is a graph illustrating median change from pre- to post-studydrug inhalation in sputum total protein levels and by drug dose level.

FIG. 3 is a graph illustrating individual sputum free elastase levelsbefore and after study drug inhalation. Slash marks represent the medianlevel.

FIG. 4 is a graph illustrating median change from pre- to post-studydrug inhalation in sputum free elastase levels and by drug dose level.

FIG. 5 is a graph illustrating individual sputum L-6 levels before andafter study drug inhalation. Slash marks represent the median level.

FIG. 6 is a graph illustrating median change from pre- to post-studydrug inhalation in sputum IL-6 levels and by drug dose level.

FIG. 7 is a graph illustrating individual sputum IL-8 levels before andafter study drug inhalation. Slash marks represent the median level.

FIG. 8 is a graph illustrating median change from pre- to post-studydrug inhalation in sputum IL-8 levels and by drug dose level.

FIG. 9 is a graph illustrating individual sputum TNF-α levels before andafter study drug inhalation. Slash marks represent the median level.

FIG. 10 is a graph illustrating median change from pre- to post-studydrug inhalation in sputum TNF-α levels and by drug dose level.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel methods for treating a pulmonarydisease state in mammals by up or down regulating indigenous in vivolevels of an inflammatory agent in mammalian cells comprising contactingthe mammalian cells with a therapeutically effective amount of aninflammatory regulator, wherein the inflammatory agent is selected fromthe group consisting of cytokines, transforming growth factor-β,elastase, and white blood cells, and wherein the inflammatory regulatoris selected from the group consisting of pyruvates and pyruvateprecursors.

As used herein, the following terms have the given meanings:

The term “injured cell” as used herein refers to a cell which has someor all of the following: (a) injured membranes so that transport throughthe membranes is diminished and may result in one or more of thefollowing, an increase in toxins and normal cellular wastes inside thecell and/or a decrease in nutrients and other components necessary forcellular repair inside the cell, (b) an increase in concentration ofoxygen radicals inside the cell because of the decreased ability of thecell to produce antioxidants and enzymes, and (c) damaged DNA, RNA andribosomes which must be repaired or replaced before normal cellularfunctions can be resumed.

The term “pharmaceutically acceptable,” such as pharmaceuticallyacceptable carriers, excipients, etc., refers to pharmacologicallyacceptable and substantially non-toxic to the subject to which theparticular compound is administered.

The term “pharmaceutically acceptable salt” refers to conventionalacid-addition salts or base-addition salts that retain the biologicaleffectiveness and properties of the compounds of the present inventionand are formed from suitable non-toxic organic or inorganic acids ororganic or inorganic bases. Sample acid-addition salts include thosederived from inorganic acids such as hydrochloric acid, hydrobromicacid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid andnitric acid, and those derived from organic acids such asp-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalicacid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid,and the like. Sample base-addition salts include those derivedfromammonium, potassium, sodium, and quaternary ammonium hydroxides,such as for example, tetramethylammonium hydroxide. Chemicalmodification of a pharmaceutical compound (i.e., drug) into a salt is atechnique well known to pharmaceutical chemists to obtain improvedphysical and chemical stability, hydroscopicity, and solubility ofcompounds. See, e.g., H. Ansel et. al., Pharmaceutical Dosage Forms andDrug Delivery Systems (6^(th) Ed. 1995) at pp. 196 and 1456-1457.

The term “prodrug” or “precursor” refers to compounds, which undergobiotransformation prior to exhibiting their pharmacological effects. Thechemical modification of drugs to overcome pharmaceutical problems hasalso been termed “drug latentiation.” Drug latentiation is the chemicalmodification of a biologically active compound to form a new compound,which upon in vivo enzymatic attack will liberate the parent compound.The chemical alterations of the parent compound are such that the changein physicochemical properties will affect the absorption, distributionand enzymatic metabolism. The definition of drug latentiation has alsobeen extended to include nonenzymatic regeneration of the parentcompound. Regeneration takes place as a consequence of hydrolytic,dissociative, and other reactions not necessarily enzyme mediated. Theterms prodrugs, latentiated drugs, and bio-reversible derivatives areused interchangeably. By inference, latentiation implies a time lagelement or time component involved in regenerating the bioactive parentmolecule in vivo. The term prodrug is general in that it includeslatentiated drug derivatives as well as those substances, which areconverted after administration to the actual substance, which combineswith receptors. The term prodrug is a generic term for agents, whichundergo biotransformation prior to exhibiting their pharmacologicalactions.

The term “therapeutically effective amount” refers to an amount of atherapeutically effective compound, or a pharmaceutically acceptablesalt thereof, which is effective to treat, prevent, alleviate orameliorate symptoms of a disease.

Pyruvates can act as inflammatory mediators (antioxidants) to neutralizeoxygen radicals directly, thus lowering the level of inflammation.Pyruvates can also act as antioxidants to regulate the synthesis ofnitric oxide. The regulation of oxygen radicals and the synthesis ofnitric oxide operate by a different set of genes than those thatregulate the synthesis of inflammatory agents such as cytokines and thusoperates by a different mechanism. Applicant has discovered thatpyruvates and pyruvate precursors can up or down regulate indigenous invivo levels of inflammatory agents such as cytokines to regulateinflammation. Specifically, applicant has discovered that pyruvates inlow dosage amounts, can down regulate the production of inflammatoryagents and the number of white blood cells to stop the negative sideeffects of chronic inflammation in uninfected pulmonary diseases or, inhigh dosage amounts, can up regulate the production of inflammatoryagents and the number of white blood cells needed to kill infections orcancer in infected pulmonary diseases. Mediation of inflammation is verydifferent from the regulation of inflammation. Mediation is a directchemical effect on the inflammatory components such as the ability ofpyruvates to act as antioxidants against oxygen radicals such ashydrogen peroxide, peroxynitrite, or nitric oxide to elicit a response.Regulation of inflammation, such as the up or down regulation of thelevels of inflammatory agents, is a direct effect of pyruvates to elicita response on a genetic level and to specifically effect and regulatethe function of inflammatory cells such as white blood cells. Theability to regulate cellular functions of inflammatory cells is verydifferent from the ability to directly chemically affect an oxygenradical. Both will lower inflammation, but only inflammatory regulatorscan up or down regulate the level of inflammation.

Pyruvates and pyruvate precursors control the positive and negativeeffects of inflammatory agents such as cytokines, transforming growthfactor-β, elastase, and white blood cells at higher levels. Too high anumber of white blood cells and other inflammatory agents is detrimentalto lungs. Pyruvates and pyruvate precursors will lower and protect cellsand organs from excess inflammatory agents and white blood cell numberswhen they are high and infections are not involved. Moderate to severeasthmatics and emphysema patients produce much higher levels ofinflammatory agents including oxygen radicals especially in smokers andlow dosages of pyruvates produce better results in these patients bylowering excess levels of inflammatory agents. The ability to controlthe levels of inflammation is important. Over production or underproduction is detrimental and produces various diseases in both thelungs and nasal cavities. Dosages of 5 ml of 0.5 mM pyruvates reduce theinflammatory markers in patients with lung diseases and can be used indiseases where inflammation is a problem, i.e., in smokers (21), mildasthmatics (21), in intubated or tracheostomized patients (19), innormal subjects after exercise and hyperventilation (21), COPD patients(22), and in patients with cystic fibrosis (22) with kartagener'ssyndrome (22), moderate or severe asthma (22), sarcoidosis (22), andfibrosing alveolitis (22). Increased levels of inflammatory cytokinesespecially IL-8, which is a neutrophil activating cytokine, arechemotactic for eosinophils, which produce and enhance inflammation(20). Acute treatment with corticosteriods during an exacerbation ofasthma is associated with a decline in inflammatory markers in adultsand children (23).

In contrast, higher dosages of pyruvates can increase the number ofwhite blood cells and the synthesis of cytokines needed in diseaseswhere cytokines are abnormally low, such as in infections. Dosages of 5ml of 5 mM pyruvates or higher beyond that needed to neutralize oxygenradicals will enter the bronchial and lung cells and increase the levelsof white blood cells and IL-1, IL-6, IL-8, TNF-α, elastase to help fightinfections. All cells have a transport system that allow cells toconcentrate pyruvate at higher concentrations than serum levels. In thecell, pyruvate raises the pH level, increases levels of ATP, decreasinglevels of ADP and cAMP, and increases levels of GTP, while decreasinglevels of cGMP.

In summary, pyruvate enhances the body's ability to reduce inflammationor to increase it to fight infections and tumors. The combination ofpyruvate alone or in combination with other drugs are effective for thetreatment of lung diseases such as asthma, emphysema etc, whereinflammation is high, and in the treatment of tumors, bacterialinfections, fungal infections, viral infections, angina, ischemicdiseases, and congestive heart failure, where inflammation is low.

The pulmonary diseases suitable for treatment by the cytokine regulatorsof the present invention include, but are not limited to, bronchialasthma, acute bronchitis, emphysema, chronic obstructive emphysema,chronic obstructive pulmonary disease, centrilobular emphysema,panacinar emphysema, chronic obstructive bronchitis, smoker's disease,reactive airway disease, cystic fibrosis, bronchiectasis, acquiredbronchiectasis, kartaagener's syndrome, acelectasis, acute atelectasis,chronic acelectasis, pneumonia, essential thrombocytemia, legionnaire'sdisease, psittacosis, fibrogenic dust disease, hypersensitivity diseasesof the lung, idiopathic infiltrative diseases of the lungs, chronicobstructive pulmonary disorder, adult respiratory distress syndrome,pulmonary tumors, and diseases caused by organic dust, irritant gases,and chemicals. Preferred disease states are cystic fibrosis, bronchialasthma, and chronic obstructive pulmonary disease.

The pulmonary tumors suitable for treatment by the cytokine regulatorsof the present invention include, but are not limited to, epidermoid(squamous cell) carcinoma, small cell (oat cell) carcinoma,adenocarcinoma, and large cell (anaplastic) carcinoma.

The inflammatory agent in the present invention may be selected from awide variety of inflammatory agents. Preferred inflammatory agents arecytokines, transforming growth factor-β, elastase, and white bloodcells. Preferred cytokines may be selected from the group consisting ofinterleukin-1, interleukin-2, interleukin-4, interleukin-6,interleukin-8, interleukin-10, interleukin-17, and interleukin-23. Morepreferred cytokines are interleukin-1, interleukin-6, and interleukin-8.IL-10, IL-17, and IL-23 are all regulated by the levels of IL-6 and IL-8and so regulation of IL-6 and IL-8 can regulate IL-10, IL-17, and IL-23.

Another preferred cytokine is tumor necrosis factor-α. Tumor necrosisfactor-α is a cytokine involved in systemic inflammation and is a memberof a group of cytokines that all stimulate the acute phase reaction.

Another preferred cytokine is interferon-α and interferon-β. Interferonsare glycoproteins that assist the immune response by inhibiting viralreplication within other cells of the body.

Another preferred inflammatory agent is transforming growth factor-β(TGF-β). Transforming growth factor-β regulates growth and proliferationof cells, blocking the growth of many different cell types includingtumor cells.

Another preferred inflammatory agent is elastase. Elastase is an enzymethat digests and degrades a number of proteins including elastin, anelastic substance found in the lungs and other organs.

Another preferred inflammatory agent is white blood cells. White bloodcells or leukocytes are cells of the immune system, which defend thebody against both infectious disease and foreign materials. Severaldifferent and diverse types of leukocytes exist, however they are allproduced and derived from a pluripotent cell in the bone marrow known asa hematopoietic stem cell. Leukocytes are found throughout the body,including the blood and lymphatic system.

The inflammatory regulators in the present invention are pyruvates andpyruvate precursors. Non-limiting illustrative examples of pyruvatesinclude pyruvic acid, lithium pyruvate, sodium pyruvate, potassiumpyruvate, magnesium pyruvate, calcium pyruvate, zinc pyruvate, manganesepyruvate, aluminum pyruvate, ammonium pyruvate, and mixtures thereof.Non-limiting illustrative examples of pyruvate precursors includepyruvyl-glycine, pyruvyl-alanine, pyruvyl-leucine, pyruvyl-valine,pyruvyl-isoleucine, pyruvyl-phenylalanine, pyruvamide, salts of pyruvicacid, and mixtures thereof.

The amount of the inflammatory regulator present in the therapeuticcompositions of the present invention is a therapeutically effectiveamount. A therapeutically effective amount of the inflammatory regulatoris that amount of the inflammatory agent necessary to treat thepulmonary disease. The exact amount of inflammatory regulator is amatter of preference subject to such factors as the type of inflammatoryregulator being employed, the type of condition being treated as well asthe other ingredients in the composition. The exact amount ofinflammatory regulator will also be determined by whether the pulmonarydisease is infected or uninfected. In general, the dosage of theinflammatory regulator may range from about 0.0001 mg to about 1 gram,preferably from about 0.001 mg to about 0.8 gram, and more preferablyfrom about 0.01 mg to about 0.6 gram.

In another embodiment, the pyruvate or pyruvate precursor inflammatoryregulator further may further comprise α-keto-isovaleric acid, or aprecursor thereof. In general, the dosage of α-keto-isovaleric acid mayrange from about 0.0001 mg to about 1 gram, preferably from about 0.001mg to about 0.8 gram, and more preferably from about 0.01 mg to about0.6 gram.

In one embodiment, the level of inflammatory agents in the mammaliancells is abnormally low in the disease state. In another embodiment, thelevel of inflammatory agents in the mammalian cells is abnormally highin the disease state.

Whether the levels of inflammatory agents that are abnormally low orabnormally high can be determined from the level of inflammatory agentsin a patient's lungs and sputum.

In many cases, pulmonary diseases produce infections that theseinflammatory regulators can treat. Such infections may be bacterial,viral, or fungal. The inflammatory regulators may be inhaled first toregulate inflammatory agents followed by inhalation of a therapeuticagent. The therapeutic agent may be administered prior to, concomitantlywith, or after administration of the inflammatory regulator. Thetherapeutic agent may be selected from the group consisting ofantibacterials, antivirals, antifungals, antitumors, antihistamines,proteins, enzymes, hormones, nonsteroidal anti-inflammatories,cytokines, nicotine, insulin, and steroids.

The antibacterial agents which may be employed in the therapeuticcompositions may be selected from a wide variety of water-soluble andwater-insoluble drugs, and their acid addition or metallic salts, usefulfor treating pulmonary diseases. Both organic and inorganic salts may beused provided the antibacterial agent maintains its medicament value.The antibacterial agents may be selected from a wide range oftherapeutic agents and mixtures of therapeutic agents, which may beadministered in sustained release or prolonged action form. Nonlimitingillustrative specific examples of antibacterial agents include bismuthcontaining compounds, sulfonamides; nitrofurans, metronidazole,tinidazole, nimorazole, benzoic acid; aminoglycosides, macrolides,penicillins, polypeptides, tetracyclines, cephalosporins,chloramphenicol, and clidamycin. Preferably, the antibacterial agent isselected from the group consisting of bismuth containing compounds, suchas, without limitation, bismuth aluminate, bismuth subcitrate, bismuthsubgalate, bismuth subsalicylate, and mixtures thereof; thesulfonamides; the nitrofurans, such as nitrofurazone, nitrofurantoin,and furozolidone; and miscellaneous antibacterials such asmetronidazole, tinidazole, nimorazole, and benzoic acid; andantibiotics, including the aminoglycosides, such as gentamycin,neomycin, kanamycin, and streptomycin; the macrolides, such aserythromycin, clindamycin, and rifamycin; the penicillins, such aspenicillin G, penicillin V, Ampicillin and amoxicillin; thepolypeptides, such as bacitracin and polymyxin; the tetracyclines, suchas tetracycline, chlorotetracycline, oxytetracycline, and doxycycline;the cephalosporins, such as cephalexin and cephalothin; andmiscellaneous antibiotics, such as chloramphenicol, and clidamycin. Morepreferably, the antibacterial agent is selected from the groupconsisting of bismuth aluminate, bismuth subcitrate, bismuth subgalate,bismuth subsalicylate, sulfonamides, nitrofurazone, nitrofurantoin,furozolidone, metronidazole, tinidazole, nimorazole, benzoic acid,gentamycin, neomycin, kanamycin, streptomycin, erythromycin,clindamycin, rifamycin, penicillin G, penicillin V, Ampicillinamoxicillin, bacitracin, polymyxin, tetracycline, chlorotetracycline,oxytetracycline, doxycycline, cephalexin, cephalothin, chloramphenicol,and clidamycin.

The amount of antibacterial agent which may be employed in thetherapeutic compositions of the present invention may vary dependingupon the therapeutic dosage recommended or permitted for the particularantibacterial agent. In general, the amount of antibacterial agentpresent is the ordinary dosage required to obtain the desired result.Such dosages are known to the skilled practitioner in the medical artsand are not a part of the present invention. In a preferred embodiment,the antibacterial agent in the therapeutic composition is present in anamount from about 0.01% to about 10%, preferably from about 0.1% toabout 5%, and more preferably from about 1% to about 3%, by weight.

The antiviral agents which may be employed in the therapeuticcompositions may be selected from a wide variety of water-soluble andwater-insoluble drugs, and their acid addition or metallic salts, usefulfor treating pulmonary diseases. Both organic and inorganic salts may beused provided the antiviral agent maintains its medicament value. Theantiviral agents may be selected from a wide range of therapeutic agentsand mixtures of therapeutic agents, which may be administered insustained release or prolonged action form. Nonlimiting illustrativecategories of such antiviral agents include RNA synthesis inhibitors,protein synthesis inhibitors, immunostimulating agents, proteaseinhibitors, and cytokines. Nonlimiting illustrative specific examples ofsuch antiviral agents include the following medicaments.

(a) Acyclovir (9-[(2-hydroxyethyloxy)methyl]guanine, ZOVIRAX®) is awhite, crystalline powder with a molecular weight of 225 Daltons and amaximum solubility in water of 2.5 mg/mL at 37° C. Acyclovir is asynthetic purine nucleoside analogue with in vitro and in vivoinhibitory activity against human herpes viruses including herpessimplex types 1 (HSV-1) and 2 (HSV-2), varicella-zoster virus (VZV),Epstein-Barr virus (EBV), and cytomegalovirus (CMV).(b) Foscarnet sodium (phosphonoformic acid trisodium salt, FOSCAVIR®) isa white, crystalline powder containing 6 equivalents of water ofhydration with an empirical formula of Na₃CO₆P₆H₂O and a molecularweight of 300.1. Foscarnet sodium has the potential to chelate divalentmetal ions such as calcium and magnesium, to form stable coordinationcompounds. Foscarnet sodium is an organic analogue of inorganicpyrophosphate that inhibits replication of all known herpes viruses invitro including cytomegalovirus (CMV), herpes simplex virus types 1 and2 (HSV-1, HSV-2), human herpes virus 6 (HHV-6), Epstein-Barr virus(EBV), and varicella-zoster virus (VZV), Foscarnet sodium exerts itsantiviral activity by a selective inhibition at the pyrophosphonatebinding site on virus-specific DNA polymerases and reversetranscriptases at concentrations that do not affect cellular DNApolymerases.(c) Ribavirin (1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide,VIRAZOLE®) is a synthetic nucleoside which is a stable, white,crystalline compound with a maximum solubility in water of 142 mg/ml at25° C. and with only a slight solubility in ethanol. The empiricalformula is C₈H₁₂N₄O₅ and the molecular weight is 244.2 Daltons.Ribavirin has antiviral inhibitory activity in vitro against respiratorysyncytial virus, influenza virus, and herpes simplex virus. Ribavirin isalso active against respiratory syncytial virus (RSV) in experimentallyinfected cotton rats. In cell cultures, the inhibitory activity ofribavirin for RSV is selective. The mechanism of action is unknown.Reversal of the in vitro antiviral activity by guanosine or xanthosinesuggests ribavirin may act as an analogue of these cellular metabolites.(d) Vidarabine (adenine arabinoside, Ara-A,9-β-D-arabinofuranosyladenine monohydrate, VIRA-A®) is a purinenucleoside obtained from fermentation cultures of Streptomycesantibioticus. Vidarabine is a white, crystalline solid with theempirical formula, C₁₀H₁₃N₅O₄H₂O. The molecular weight of vidarabine is285.2, the solubility is 0.45 mg/ml at 25° C., and the melting pointranges from 260° C. to 270° C. Vidarabine possesses in vitro and in vivoantiviral activity against Herpes simplex virus types 1 and 2 (HSV-1 andHSV-2), and in vitro activity against varicella-zoster virus (VZV). Theantiviral mechanism of action has not yet been established. Vidarabineis converted into nucleotides, which inhibit viral DNA polymerase.(e) Ganciclovir sodium (9-(1,3-dihydroxy-2-propoxymethyl)guanine,monosodium salt, CYTOVENE®, CYMEVENE®) is an antiviral drug activeagainst cytomegalovirus for intravenous administration. Ganciclovirsodium has a molecular formula of C₉H₁₂N₆NaO₄ and a molecular weight of277.21. Ganciclovir sodium is a white lyophilized powder with an aqueoussolubility of greater than 50 mg/mL at 25° C. Ganciclovir is a syntheticnucleoside analogue of 2′-deoxyguanosine that inhibits replication ofherpes viruses both in vitro and in vivo. Sensitive human virusesinclude cytomegalovirus (CMV), herpes simplex virus-1 and -2 (HSV-1,HSV-2), Epstein-Barr virus (EBV), and varicella zoster virus (VZV).(f) Zidovudine [azidothymidine (AZT), 3′-azido-3′-deoxythymidine,RETROVIR®] is an antiretroviral drug active against humanimmunodeficiency virus (HIV) for oral administration. Zidovudine is awhite to beige, odorless, crystalline solid with a molecular weight of267.24 Daltons and a molecular formula of C₁₀H₁₃N₅O₄. Zidovudine is aninhibitor of the in vitro replication of some retroviruses including HIV(also known as HTLV III, LAV, or ARV). Zidovudine is a thymidineanalogue in which the 3′-hydroxy (—OH) group is replaced by an azido(—N₃) group.(g) Phenol (carbolic acid) is a topical antiviral, anesthetic,antiseptic, and antipruritic drug. Phenol is a colorless or whitecrystalline mass, which is soluble in water, has a characteristic odor,a molecular formula of C₆H₆O, and a molecular weight of 94.11.(h) Amantadine hydrochloride (1-adamantanamine hydrochloride,SYMMETREL®) has pharmacological actions as both an anti-Parkinson and anantiviral drug. Amantadine hydrochloride is a stable white or nearly,white crystalline powder, freely soluble in water and soluble in alcoholand in chloroform. The antiviral activity of amantadine hydrochlorideagainst influenza A is not completely understood but the mode of actionappears to be the prevention of the release of infectious viral nucleicacid into the host cell.(i) Interferon α-n3 (human leukocyte derived, ALFERON®) is a sterileaqueous formulation of purified, natural, human interferon α proteinsfor use by injection. Interferon α-n3 injection consists of interferon aproteins comprising approximately 166 amino acids ranging in molecularweights from 16,000 to 27,000 Daltons. Interferons are naturallyoccurring proteins with both antiviral and antiproliferative properties.

(j) Interferon α-2a (recombinant, ROFERON-A®) is a sterile proteinproduct for use by injection. Interferon α-2a is a highly purifiedprotein containing 165 amino acids, and it has an approximate molecularweight of 19,000 Daltons. The mechanism by which Interferon α-2a,recombinant, exerts antitumor or antiviral activity is not clearlyunderstood. However, it is believed that direct antiproliferative actionagainst tumor cells, inhibition of virus replication, and modulation ofthe host immune response play important roles in antitumor and antiviralactivity.

(k) Oseltamivir ((3R,4R,5S)-4-acetylamino-5-amino-3-(1-ethylpropoxy)-1-cyclohexene-1-carboxylicacid ethyl ester, TAMIFLU®) is a is an antiviral drug that is used inthe treatment and prophylaxis of both influenza virus A and Influenzavirus B. Oseltamivir is a neuraminidase inhibitor. It acts as atransition-state analogue inhibitor of influenza neuraminidase,preventing new viruses from emerging from infected cells. Oseltamivirhas a molecular formula of C₁₆H₂₈N₂O₄.

Preferred antiviral agents to be employed may be selected from the groupconsisting of acyclovir, foscarnet sodium, ribavirin, vidarabine,ganciclovir sodium, zidovudine, phenol, amantadine hydrochloride, andinterferon α-n3, interferon α-2a, and oseltamivir. In a preferredembodiment, the antiviral agent is selected from the group consisting ofacyclovir, foscarnet sodium, ribavirin, vidarabine, and ganciclovirsodium. In a more preferred embodiment, the antiviral agent isacyclovir.

The amount of antiviral agent which may be employed in the therapeuticcompositions of the present invention may vary depending upon thetherapeutic dosage recommended or permitted for the particular antiviralagent. In general, the amount of antiviral agent present is the ordinarydosage required to obtain the desired result. Such dosages are known tothe skilled practitioner in the medical arts and are not a part of thepresent invention. In a preferred embodiment, the antiviral agent in thetherapeutic composition is present in an amount from about 0.1% to about20%, preferably from about 1% to about 10%, and more preferably fromabout 2% to about 7%, by weight.

The antifungal agents which may be employed in the therapeuticcompositions may be selected from a wide variety of water-soluble andwater-insoluble drugs, and their acid addition or metallic salts, usefulfor treating pulmonary diseases. Both organic and inorganic salts may beused provided the antifungal agent maintains its medicament value. Theantifungal agents may be selected from a wide range of therapeuticagents and mixtures of therapeutic agents, which may be administered insustained release or prolonged action form. Nonlimiting illustrativespecific examples of antifungal agents include the followingmedicaments: miconazole, clotrimazole, tioconazole, terconazole,povidone-iodine, and butoconazole. Other antifungal agents are lacticacid and sorbic acid. Preferred antifungal agents are miconazole andclotrimazole.

The amount of antifungal agent, which may be employed in the therapeuticcompositions of the present invention may vary depending upon thetherapeutic dosage recommended or permitted for the particularantifungal agent. In general, the amount of antifungal agent present isthe ordinary dosage required to obtain the desired result. Such dosagesare known to the skilled practitioner in the medical arts and are not apart of the present invention. In a preferred embodiment, the antifungalagent in the therapeutic composition is present in an amount from about0.05% to about 10%, preferably from about 0.1% to about 5%, and morepreferably from about 0.2% to about 4%, by weight.

The antitumor agents which may be employed in the therapeuticcompositions may be selected from a wide variety of water-soluble andwater-insoluble drugs, and their acid addition or metallic salts, usefulfor treating pulmonary diseases. Both organic and inorganic salts may beused provided the antitumor agent maintains its medicament value. Theantitumor agents may be selected from a wide range of therapeutic agentsand mixtures of therapeutic agents, which may be administered insustained release or prolonged action form. Nonlimiting illustrativespecific examples include anti-metabolites, antibiotics, plant products,hormones, and other miscellaneous chemotherapeutic agents. Chemicallyreactive drugs having nonspecific action include alkylating agents andN-alkyl-N-nitroso compounds. Examples of alkylating agents includenitrogen mustards, azridines (ethylenimines), sulfonic acid esters, andepoxides. Anti-metabolites are compounds that interfere with theformation or utilization of a normal cellular metabolite and includeamino acid antagonists, vitamin and coenzyme antagonists, andantagonists of metabolites involved in nucleic acid synthesis such asglutamine antagonists, folic acid antagonists, pyrimidine antagonists,and purine antagonists. Antibiotics are compounds produced bymicroorganisms that have the ability to inhibit the growth of otherorganisms and include actinomycins and related antibiotics, glutarimideantibiotics, sarkomycin, fumagillin, streptonigrin, tenuazonic acid,actinogan, peptinogan, and anthracyclic antibiotics such as doxorubicin.Plant products include colchicine, podophyllotoxin, and vinca alkaloids.Hormones include those steroids used in breast and prostate cancer andcorticosteriods used in leukemias and lymphomas. Other miscellaneouschemotherapeutic agents include urethane, hydroxyurea, and relatedcompounds; thiosemicarbazones and related compounds; phthalanilide andrelated compounds; and triazenes and hydrazines. The anticancer agentmay also be a monoclonal antibody or the use of X-rays. In a preferredembodiment, the anticancer agent is an antibiotic. In a more preferredembodiment, the anticancer agent is doxorubicin. In a most preferredembodiment, the anticancer agent is doxorubicin.

The amount of antitumor agent, which may be employed in the therapeuticcompositions of the present invention may vary depending upon thetherapeutic dosage recommended or permitted for the particular antitumoragent. In general, the amount of antitumor agent present is the ordinarydosage required to obtain the desired result. Such dosages are known tothe skilled practitioner in the medical arts and are not a part of thepresent invention. In a preferred embodiment, the antitumor agent in thetherapeutic composition is present in an amount from about 1% to about50%, preferably from about 10% to about 30%, and more preferably fromabout 20% to about 25%, by weight.

Nicotine is an alkaloid found predominantly in tobacco and constitutesabout 0.6-3% of tobacco by dry weight. In low concentrations, an averagecigarette yields about 1 mg of absorbed nicotine. Nicotine acts as astimulant in mammals and is one of the main factors responsible for thedependence-forming properties of tobacco smoking. Nicotine is ahygroscopic, oily liquid that is miscible with water in its base form.As a nitrogenous base, nicotine forms salts with acids that are usuallysolid and water-soluble. The primary therapeutic use of nicotine is intreating nicotine dependence in order to eliminate smoking with itshealth risks.

Insulin is an animal hormone, produced in the pancreas, whose presenceinforms the body's cells that the animal is well fed, causing liver andmuscle cells to take in glucose and store it in the form of glycogen,and causing fat cells to take in blood lipids and turn them intotriglycerides. Insulin is used medically to treat some forms of diabetesmellitus. Patients with type 1 diabetes mellitus depend on externalinsulin (most commonly injected subcutaneously) for their survivalbecause of the absence of the hormone. Patients with type 2 diabetesmellitus have insulin resistance, relatively low insulin production, orboth; some type 2 diabetics eventually require insulin when othermedications become insufficient in controlling blood glucose levels.Insulin is a peptide hormone composed of 51 amino acid residues.Insulin's genetic structure varies marginally between species of animal.Bovine insulin differs from human in only three amino acid residues, andporcine insulin in one. Even insulin from some species of fish issimilar enough to human to be effective in humans. The C-peptide ofproinsulin, however, is very divergent from species to species. Allstructures of insulin useful in humans, including synthetic “human”insulin, may be employed in the present invention. Unlike manymedicines, insulin cannot be taken orally. Like nearly all proteinsintroduced into the gastrointestinal tract, insulin is degraded losingall insulin activity. Insulin is usually taken as subcutaneousinjections or may be inhaled.

The carrier composition is selected from the group consisting oftablets, capsules, liquids, isotonic liquids, isotonic media, enterictablets and capsules, parenterals, topicals, creams, gels, ointments,chewing gums, confections and the like. The favored method of deliveryis through inhalation by mouth or sinuses.

Obviously, numerous modifications and variations of the presentinvention are possible in the light of the above teachings and theinvention is not limited to the examples herein. It is thereforeunderstood that within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described herein.

Throughout this application, various publications have been referenced.The disclosures in these publications are incorporated herein byreference in order to more fully describe the state of the art.

The compounds of the present invention can be prepared according to theexamples set out below. The examples are presented for purposes ofdemonstrating, but not limiting, the preparation of the compounds andcompositions of this invention.

EXAMPLES Example 1 Inhaled Sodium Pyruvate for the Treatment of CysticFibrosis Double Blind, Placebo-Controlled, Safety Study SputumInflammatory Biomarkers

All of the enrolled and dosed subjects were able to provide sputumsamples for analysis before and after exposure to study drug (sodiumpyruvate for inhalation at 0.5, 1.5, and 5.0 mM levels). The subjectswere given 5 ml samples to inhale. Specimens were of good quality forthe planned assays.

The 0.5 mM levels of sodium pyruvate using 5 ml samples contain 0.28 mgof sodium pyruvate. The 5 mM levels of sodium pyruvate using 5 mlcontain 2.8 mg of sodium pyruvate.

Samples were divided into two main aliquots after processing. The firstaliquot was left untreated to be able to assay for the activity of freeelastase. The second aliquot was treated with protease inhibitorspheylmethanesulfonylfluoride (PMSF) and ethylenediamine tetraacetic acid(EDTA) to stop any degradation of the cytokines of interest (IL-6, IL-8,IL-10, IL-17, and IL-23) and total protein.

For IL-10, IL-17 and IL-23, levels on sputum were at or below the limitof detection for the assays (7, 2 and 20 pg/mL respectively). For theother markers (total protein, elastase, IL-6, IL-8, TNF-α) levelsdetected where within those typically found in cystic fibrosis (CF)patients. Overall, significant changes were noted in these biomarkerstested in sputum by drug dose level or for the group as a whole (FIGS.1-10).

FIG. 1 is a graph illustrating individual sputum total protein levelsbefore and after study drug inhalation. Slash marks represent the medianlevel.

FIG. 2 is a graph illustrating median change from pre- to post-studydrug inhalation in sputum total protein levels and by drug dose level.

FIG. 3 is a graph illustrating individual sputum free elastase levelsbefore and after study drug inhalation. Slash marks represent the medianlevel.

FIG. 4 is a graph illustrating median change from pre- to post-studydrug inhalation in sputum free elastase levels and by drug dose level.

FIG. 5 is a graph illustrating individual sputum IL-6 levels before andafter study drug inhalation. Slash marks represent the median level.

FIG. 6 is a graph illustrating median change from pre- to post-studydrug inhalation in sputum IL-6 levels and by drug dose level.

FIG. 7 is a graph illustrating individual sputum IL-8 levels before andafter study drug inhalation. Slash marks represent the median level.

FIG. 8 is a graph illustrating median change from pre- to post-studydrug inhalation in sputum IL-8 levels and by drug dose level.

FIG. 9 is a graph illustrating individual sputum TNF-α levels before andafter study drug inhalation. Slash marks represent the median level.

FIG. 10 is a graph illustrating median change from pre- to post-studydrug inhalation in sputum TNF-α levels and by drug dose level.

Given the changes noted in the cell counts (both in peripheral blood andsputum) this finding is intriguing. Since evidence for cellular influxwas noted, it would have been expected to see this paralleled by acorresponding increase in cytokines and particularly in the freeelastase activity. The study drug blocked this pro-inflammatory effect.The data clearly showed that pyruvate can up or down regulateinflammation depending on concentration. White blood cell counts werereduced 25% with the inhalation of 5 ml of 0.5 mM sodium pyruvate, aswas total proteins, elastase, as were IL-6, IL-8, and TNF-α. White bloodcell counts were increased by 25% with the inhalation of 5 ml of 5 mMpyruvate or higher as was the total proteins, elastase, IL-6, IL-8, andTNF-α.

Tissue Culture Studies

To investigate the ability of pyruvate to regulate the inflammatoryprocess during an infection, the MatTek EpiDerm Assay was used. TheMatTek Epiderm tissue samples were treated with pyruvate and thecombination of pyruvate and α-ketoisovalerate both at 20 mMconcentrations or higher to determine if the combination would regulateIL-1 and IL-8 up or down during a simulated infection. Following aone-hour equilibration, the Epiderm tissues were placed into theincubator (37° C., 5% CO₂) in assay medium. The old medium was replacedwith fresh medium and the test articles were applied to the tissuesamples. The test articles remained in contact with the tissue forvarious dosing times, one hour, then at four hours, and at 20 hours. Thetesting was run in duplicate. Various immunostimulators sodium dodecylsulfate (SDS), glycoproein D (gpD) were used singly or with the α-ketoacids to replicate an infection, along with vehicle controls. Untreatedsamples were used as negative controls. Following treatment, the mediafrom the tissues samples were tested in Elisa kits for IL-1 and IL-8according to the manufacture's protocols.

The 0.5 mM levels of sodium pyruvate using 5 ml samples contain 0.28 mgof sodium pyruvate. The 10 mM levels of sodium pyruvate using 5 mlcontain 5.6 mg of sodium pyruvate. The 20 mM levels of sodium pyruvateusing 5 ml contain 11.2 mg of sodium pyruvate. The 40 mM levels ofsodium pyruvate using 5 ml contain 22.4 mg of sodium pyruvate.

A quantity of 5 ml of 0.1 mM to 100 mM of α-keto isovalerate was used. Aquantity of 5 ml of 20 mM of α-keto isovalerate contains 13.8 mg. Aquantity of 5 ml of 40 mM of α-keto isovalerate contains 27.6 mg. Aquantity of 5 ml of 100 mM of α-keto isovalerate contains 69 mg.

Results

The primary end points were the levels of IL-8 and IL-1 after treatmentwith an immunostimulator, pyruvate and the combination of pyruvate andα-ketoisovalerate. The immunostimulator did not increase the cytokinesby themselves. This model did not have white blood cells to respond tothe immunostimulator or produce oxygen radicals. The α-keto acids didnot increase the cytokines also in this model. The immunostimulators incombination with pyruvate and α-ketoisovalerate increased IL-8 over300%, which shows direct antimicrobial activity, compared to theuntreated controls. IL-8 activates neutrophils to increase their numbersat the infected site. In the same experiment, IL-1 was decreasedsignificantly (over 200%). IL-1 increases inflammation and decreaseshealing times. This test clearly showed that the α-keto acids regulatedthe inflammatory process in dermal tissues in a manner that wouldincrease the bodies ability to fight infected wounds and increase thebody's ability to healing quicker. The same experiment was done withvirally infected cells and the pyruvate and combination of pyruvate andα-ketoisovalerate decreased viral plaque formation by 50%. Viral plaquesare a direct measure of viral numbers in infected cells. The antiviraldrug, Acyclovir also decreased viral plaques by 60% and the α-keto acidsin combination with acyclovir, totally eliminated the virus from theinfected cells.

Example 2 Inhaled Sodium Pyruvate for the Treatment of Cystic Fibrosisand Other Lung Diseases Clinical Trials

Cystic fibrosis (CF) is the most common, lethal inherited disease ofCaucasians. Approximately 30,000 people in the United States and 70,000worldwide have a diagnosis of CF. It is caused by mutations in thecystic fibrosis transmembrane regulator (CFTR) gene. The clinicalmanifestations characteristic of CF include progressive bronchiectaticlung disease with thick mucus production and colonization by Pseudomonasaeruginosa. The CFTR gene mutation results in altered cell transportproperties, which affect both chloride and glutathione secretion.Chronic inflammation, associated with activated neutrophils andmacrophages, is a common feature of CF. Highly reactive toxic oxygen(superoxide anion, free hydroxyl required hydrogen peroxide) andnitrogen species (NO peroxynitrites) are abundant in the chronicinflammatory response in CF and appear to playa prominent role in thepathogenesis of this disease as are excess levels of inflammatorycytokines.

A total of 15 CF patients were treated with varying doses of sodiumpyruvate. The therapeutic dose, 0.5 mm lowered the inflammatorycytokines (markers) including, IL-6, IL-8 and the 5 mm sodium pyruvateincreased all the inflammatory markers. The 0.5 mm can be used in CFpatients to lower inflammation and white blood cell numbers and the 5 mmcan be used to increase cytokines and white blood cell numbers needed tofight infections.

Treatment of HSV-I Infected Cell with Various α-Keto Acids α-Keto AcidsRegulation of Inflammatory Anti Viral Cytokines at Varying MmConcentrations α-Keto Acids Tested Alone for their Ability to ReduceViral Plaques (Numbers Live Viruses) Percentage of Viral PlaqueReduction in Virally Infected Cells

pyruvate + pyruvate + α-keto α-keto α-keto α-keto pyruvate isovaleratebutyrate isovalerate butyrate 05 mM  5%  0% 0% 10%  0% 10 mM 10%  5% 0%20%  3% 20 mM 38% 10% 0% 50% 30% 40 mM 50% 20% 5% 74% 40%

α-Keto Acids in Combination with Acyclovir (Therapeutic Dose) Percentageof Viral Plaque Reduction in Virally Infected Cells (Reduction of LiveViruses)

α-keto Pyruvate isovalerate α-keto butyrate therapeutic dose AcyclovirAcyclovir Acyclovir Acyclovir alone 10 mM 66% 42% 39% 40% 20 mM 90% 55%40%

α-Keto Acids Combinations with Acyclovir Percentage of Viral PlaqueReduction in Virally Infected Cells

Pyruvate Pyruvate α-keto isovalerate α-keto butyrate Acyclovir Acyclovir10 mM 78% 63% 20 mM 100% 70%

The results clearly show that pyruvate was the only α-keto acid thatincreased the inflammatory cytokines high enough needed to kill highnumbers of the virus in virally infected cells as measured by reductionin viral plaques. Acyclovir a known anti-viral agent, did reduce viralplaques also. Unexpectedly, the combination of pyruvate and α-ketoisovalerate produced the best results, by totally eliminating the virusfrom the infected cells. Inhalation of pyruvate at 0.5 mM reduced thelevels of white blood cells, elastase, IL-8, IL-6 and did not increaselevels of TNF-α. Inhalation of 5 mM of pyruvate in humans increasedlevels of white blood cells elastase, IL-8, IL-6 and TNF-α (cytokines),that would be used to kill viruses in the lungs. This tissue culturedata along with data from humans, confirms that some α-keto acids workedand some like α-keto butyrate did not. It appears that α-keto butyratewill reduce inflammation and the production of cytokines, even during aninfection.

REFERENCES

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While a number of embodiments of this invention have been represented,it is apparent that the basic construction can be altered to provideother embodiments that utilize the invention without departing from thespirit and scope of the invention. All such modifications and variationsare intended to be included within the scope of the invention as definedin the appended claims rather than the specific embodiments that havebeen presented by way of example.

1. A method for treating a pulmonary and upper respiratory disease state in mammals, said disease state characterized by abnormally low levels of inflammatory agents, said method comprising: contacting the mammalian cells with an inflammatory regulator comprising at least 2.8 mg pyruvate, a pyruvate precursor, or a salt thereof; wherein the inflammatory agents which are at abnormally low levels are selected from the group consisting of elastase, white blood cells, and cytokines selected from the group consisting of interleukin-6, interleukin-8, interleukin-10, interleukin-17, interleukin-23 and tumor necrosis factor-α; wherein the disease state is chronic obstructive pulmonary disease (COPD).
 2. The method according to claim 1, wherein the cytokine is selected from the group consisting of interleukin-6, interleukin-8, interleukin-10, interleukin-17, and interleukin-23.
 3. The method according to claim 2, wherein the cytokine is selected from the group consisting of interleukin-6, and interleukin-8.
 4. The method according to claim 1, wherein the cytokine is tumor necrosis factor-α.
 5. The method according to claim 1, wherein the inflammatory agent is selected from the group consisting of elastase, and white blood cells.
 6. The method according to claim 1, wherein the pyruvate is selected from the group consisting of pyruvic acid, lithium pyruvate, sodium pyruvate, potassium pyruvate, magnesium pyruvate, calcium pyruvate, zinc pyruvate, manganese pyruvate, aluminum pyruvate, ammonium pyruvate, and mixtures thereof.
 7. The method according to claim 1, wherein the pyruvate precursors are selected from the group consisting of pyruvyl-glycine, pyruvyl-alanine, pyruvyl-leucine, pyruvyl-valine, pyruvyl-isoleucine, pyruvyl-phenylalanine, pyruvamide, salts of pyruvic acid, and mixtures thereof.
 8. The method according to claim 1, wherein the inflammatory regulator contains from about 2.8 mg to about 1 gram of pyruvate, a pyruvate precursor, or a salt thereof.
 9. The method according to claim 1 further comprising contacting the mammalian cells with a therapeutic agent.
 10. The method according to claim 9, wherein the therapeutic agent is a steroid.
 11. The method according to claim 9, wherein the therapeutic agent is administered prior to administration of the inflammatory regulator.
 12. The method according to claim 9, wherein the therapeutic agent is administered concomitantly with administration of the inflammatory regulator.
 13. The method according to claim 9, wherein the therapeutic agent is administered after administration of the inflammatory regulator. 